ABSTRACT
Pinus densiflora needle extract (PDNE) is widely reported to have many pharmacological activities including antioxidant potential. However, the solvent system used for extraction greatly affects its antioxidant quality. Hence, in the present study, we investigated the effect of a different ratio (vol/vol) of ethanol to water (0-100％) in the extraction of PDNE with potent antioxidant capacity. The chemical assays, 2,2-diphenyl-1 picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), were conducted to assess the antioxidant potential of PDNE. Subsequently, the cytoprotective effect of PDNE was determined using tert-butyl hydroperoxide (TBHP)-challenged HepG2 cellular model. The needle extracts from 40％ ethanol (PDNE-40) showed greater radical scavenging activity followed by 60％, 20％, 80％, 0％ and 100％ ethanol extracts. EC50 value of the most active extract, PDNE-40, was 8.56 ± 0.51 μg/mL, relative to 1.34 ± 0.28 μg/mL of the standard trolox (for ABTS radical), and 75.96 ± 11.60 μg/mL, relative to 4.83 ± 0.26 μg/mL of the standard trolox (for DPPH radical). Either PDNE-20 or PDNE-40 pretreatment remarkably decreased the levels of reactive oxygen species (ROS), lipid peroxides and protein carbonyls in TBHP-challenged HepG2 cells. In addition, both PDNE-20 and PDNE-40 significantly reversed the decreased ratio of reduced (GSH) to oxidized (GSSG) glutathione. Moreover, these two extracts showed a significant inhibitory effect on TBHP-induced nuclear damage and loss of cell viability. In summary, the inclusion of 40％ ethanol in water for extraction of Pinus densiflora needle greatly increases the antioxidant quality of the extract.
ABSTRACT
Acanthopanax sessiliflorus, a small woody shrub has traditionally been referred to have anticancer activity, but it has not been scientifically explored so far. Therefore, to investigate the anticancer effects of A. sessiliflorus stem bark extracts (ASSBE), MDA-MB-231 and MCF-7 human breast cancer cells were treated with one of its bioactive fractions, n-hexane (ASSBE-nHF). Cytotoxicity (24 h) was determined by MTT assay and antiproliferative effect was assessed by counting cell numbers after 72 h treatment using hemocytometer. The role of ASSBE-nHF on apoptosis was analysed by annexin V-FITC/PI, Hoechst 33342 staining, DNA fragmentation pattern and immunoblotting of apoptosis markers. For the assay of enhanced production of ROS and mitochondrial membrane depolarization, specific stains such as DCFH-DA and JC-1 were used, respectively. To understand the mode of action of ASSBE-nHF on MCF-7 cells, cells were pre-treated with antioxidant, n-acetylcysteine. The hexane fraction of ASSBE showed maximum activity towards human breast cancer cells compared to other two fractions at a minimal concentration of 50 µg/ml. The annexin V-FITC/PI, Hoechst 33342 staining, DNA fragmentation and immunoblotting assays showed that ASSBE-nHF induces non-apoptotic cell death in MCF-7 and MDA-MB-231 cells. ASSBE-nHF significantly increased the production of ROS and decreased the mitochondrial membrane potential (MMP) in MCF-7 cells. Similarly, it decreased the MMP in MDA-MB-231 cells, but had no effect on ROS production. Further, the cytotoxic effect of ASSBE-nHF in MCF-7 cells was not significantly reversed even in the presence of n-acetylcysteine, an antioxidant. These findings revealed that ASSBE-nHF induces non-apoptotic cell death via mitochondria associated with both ROS dependent and independent pathways in human breast cancer cells.